The goal of the proposal is to design and synthesize modified nucleotides to increase their size difference for single molecule DNA analysis by nanopores. We will pursue the following aims to study the feasibility of this approach: (1) Use solid phase synthesis to prepare single stranded DNA consisting of nucleotides carrying different sized modification groups and test these modified DNAs using nanopores to evaluate the parameters that are required to generate distinct blockade signals from each nucleotide in the DNA; (2) With knowledge gained in aim 1, design and synthesize modified nucleotides carrying different size groups for synthesis of modified DNAs in polymerase reaction. The single stranded DNA will then be detected using nanopores to search for condition to guide the design and modification of the nucleotides to achieve distinct blockade signals; (3) Design and synthesize nucleotides carrying small functional groups as hooks for DNA polymerase reaction to generate hook-labeled DNA products. Due to the small size of the hook, these nucleotides are expected to be good substrates for commonly used DNA polymerase to produce DNA products carrying the hook. The single stranded DNA products carrying the hook will then be isolated and selectively reacted with several different large functional groups to increase the size difference among the nucleotides in DNA. This DNA strand with the modified nucleotides will then be detected distinctly by nanopores to produce sequence data. The molecular tools developed here will facilitate achieving the long-term goal of single molecule sequencing by nanopores at single base resolution. [unreadable] [unreadable]